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Evaluation of living Gumboro vaccineprepared from locally variant isolated strain for controlling of IBD problem in Egypt *Susan, S. El-mahdy, ** Manal. A. Afify, **Abd Alim, G., and *Helal, A. M. (*) Central Lab. For Evaluation of Veterinary Biologics (CLEVB) (**) Faculty of Vet.Medicine, Cairo Uni. [email protected]. Abstract: For controlling of Gumboro problem in Egypt; the locally isolated variant strain was adapted on BGM-70 cell line used for preparation of tissue culture living vaccine. The IBDV had a titer of 107.5 TCID50/ml (104.5 TCID50 per dose) after five passages in BGM-70 cell culture. Evaluation of prepared living variant vaccine was measured by identity,purity, safety and potency. The result revealed that the prepared vaccine was free from bacterial (aerobic-anaerobic), fungal mycoplasmal and extraneous viral contamination. The protection percentage was 92%, 96% and 100% in groups vaccinated with imported vaccine compared to 96% in group vaccinated with local prepared vaccine when challenged with vvIBDV. On the other hand, the protection percentage with significant higher in groups challenged with IBD variant strain. Finally, protective dose fifty for locally prepared variant vaccine were significant in full and half dose. Keywords: Local variant Infectious bursal disease virus (IBDV)-Tissue culture living vaccine(T.C) Introduction: Gumboro disease (IBD) was described as a specific new disease by Cosgrove (1962) and was referred to as “avian nephrosis”. Later, it was termed as infectious bursal disease by Hitchner (1970). It is acute highly contagious viral disease of young chickens characterized by enlargement of the bursa of Fabricius and sever renal damages (Ivanyi and Morris, 1976). Acute IBD emerged in 1980s (Jackwood, et al., 1982). IBD was first reported in Egyptian flocks in the early seventies (El-Sergany et al., 1974). However interest in IBDV antigenic characterization was triggered by the appearance of the very virulent IBD in vaccinated Egyptian flocks (El-Batrawi and El-Kady 1990;Khafagy, et al.,1991).Several reports have classified the Egyptian IBDV isolates as classical IBDV (Khafagy, et al., 1991; Bekhit, 1996; Ibrahim, 2000). Presently, evidence of circulating variant IBDV strains were isolated from flocks vaccinated using classical IBDV vaccines (El-Khiat, 2003; Hussein et al., 2003 and Metwally et al., 2003).Variant strains of IBDV are usually isolated from vaccinated flocks. These IBDV variants are antigenically different from classic strains of IBDV; as it is devoid the classical epitope(s) defined by neutralizing monoclonal antibodies (Snyder et al., 1988b; Snyder, 1990; Snyder et al., 1992).Most of these epitopes are located in the VP2 hyper variable region (Snyder et al., 1988a). Immunization of chickens is the principle method used for the control of IBD in chickens. There are many choice of available live vaccine based on virulence as intermediate virulence and highly attenuated strains while virulent vaccine not available commercially up till now, also the vaccines can be classified according to antigenic variation. The vaccine must be safe, pure and efficient (Tsukamoto et al., 1995).Inspire of vaccination against IBD, some flocks were suffered from immunosuppression .As well as some flocks up to 3weeks (unsusceptible age of classical IBD) were immunosuppressed with atrophied bursae and lesion of proventriculus. The aim of this study was carried out to controlling IBD problem in Egypt by preparation of T.C vaccine from the isolated variant strain and possibility of cross protection between variant and classical one. Material and Methods: Viral strains: 1. Infectious bursal disease viruses (IBDV): 1.1-Field isolated variant viruses: Infectious bursal disease virus isolate Egy-IBD Var 2009 VP2 gene, partial cds Submitted in gen bank at Accession No. : JN 118617. -1- 1.2-Challenge(very virulent (VVIBD)and classic) viruses: The viruses used in the challenge were in form of infectious allantoic fluid, they were isolated from field cases and identified by phylogenic analysis .They were titrated in SPF ECE as described by Villegas (1990) with titer103.5 EID50/ml and it is calculated according to the method of Reed and Muench (1938). 2. Newcastle disease viruses (NDV): 2.1-Newcastle disease challenged virus: It is a virulent virus of Newcastle disease of field isolate, it was obtained from the Newcastle Disease Department, Veterinary Serum and Vaccine Research Institute, Abbassia, Cairo (VSVRI), Its infectivity titer was106 EID50/ml.It used for challenge the experimental chicks 3 weeks post vaccination. 2.2-Newcastle diseaseheamagglutinating antigen: Propagation of LaSota strain NDV in embryonated chicken egg was applied according to Allan et al., (1972) for HI test. ND Heamagglutinating antigen was adjusted 4 HA unite. 3. Vaccine strains: 3.1- Infectious bursal disease (IBD)vaccines: Four living attenuated imported gumboro vaccine were used: Intermediate plus obtained from CEVA with batch no. 3412T3S, A CevaIBD2; with titer105.5 EID50 / dose. Intermediate obtained from IZO with batch no. 0018E IZO Gumboro 2,with titer 105.5 EID50 / dose Intermediate obtained from Intervet International B.V.,Box Meer, Holland with batch no. 09614HMol NobilisGumboro 228E;with titer 105.5 EID50 / dose Classical D78obtained from Hipra with batch no.9R2y-2 M.G. Hipra, with titer 105.2 EID50 / dose. 3.2- Newcastle Disease vaccine: (vaccination of experimental chicks for evaluation of immunosuppression effect of IBD vaccine) NDV Hitchner B1 vaccinal strain obtained from Intervet B.V., Box Meer Holland with batch no. 08811EJ01 Nobilis ND Hitchner. This vaccine used for immunosuppression study of IBD with titer 107.5EID50/ml. 4. Experimental hosts: 4.1. Experimental chicks: One day old specific pathogen free (SPF) chicks were used in this study. All birds were reared in separated cages and kept in strictly hygienic measures. The room was previously cleaned, thoroughly disinfected and were provided with autoclaved commercial water and feed. The chicks used for evaluation of prepared vaccine study. 4.2. Specific pathogen free (SPF) embryonating chicken eggs (ECE): It was obtained from the SPF production farm, Koum Oshein, El-Fayoum, Egypt. Eggs were kept in the egg incubator at 37 °C with humidity 40-60% used according to Allan et al., ( 1972) for detection of extraneous haemagglutinating agents in prepared vaccine. 4.3. Experimental mice: Swiss male mice were obtained from (CLEVB), used for studying the Safety of T.C IBD vaccine for mammalian species. (Ecotoxicity test) 5. Tissue cultures and cell culture media: (Lennelte, 1964) Primary chicken embryo fibroblast cell (CEF) was obtained from (CLEVB); which prepared as -2- described by Schat and Purchase (1989). Trypsin-version solution prepared according to Lennelte (1964); Hank's balanced salt solution (HBSS) prepared according to Hank andwallance (1949); Minimum Essential Medium (MEM) was prepared according to the manufacturer’s instructions; and Bovine serum was mycoplasma free and virus screened "Gibco Limited, Scotland, and UK". The method used for inoculation in the microtitre plates was done according to Rossiter and Jessett (1982) Tissue culture used for detection of extraneous agents in prepared IBD vaccine (Avian Lymphoid Leukosis) according to 9 CFR Ch. 1 (1-1-09 Edition) 11.30 (2009). Baby Grivet Monkey Kidney cell line 70 (BGM-70) were used forvariant virus propagation and titration according to the method described by Jackwood et al., (1986). It was obtained from Vacciera, Agouza,Giza 6. Serum samples: Sera were collected from the tested chickens three weeks after experimental infection (post inoculation) and for the evaluation of the immune response of both prepared and imported IBD vaccine stored at -20°C till used. Methods: 1. Propagation of local field isolates in BGM-70 cell lines: According to Jackwood et al. (1986). 2. Enzyme Linked Immuno-Sorbent Assay (ELISA): (Snyder et al.,1986). ELISA Kit was obtained from Symbiotic Corporation 11011 VIA Forntera San Digo, CA 92127, U.S.;Leukosis ELISA Kit with Batch No. FS 5254 and Infactiousbursal kit with Batch no FS5155.ELISA Reader :Micro plate reader USA,VERSA max,with serial number was B02274. 3. Polymerase Chain reaction (PCR): (Council of Europe, 1999). PCR used for test of extraneous agents in IBD prepared vaccine according to Council of Europe (1999)and for detect of Identity of IBD Vaccine by using DNA Star analysis; RNA extraction Kit using Bioflux simply total RNA extraction Kit Cat # BSC 52 S1 ). DNA extraction Kit using Bioflux Mega Bio virus DNA purification kit cat # BSC 12 S14. Amplification by using BIOER Reverse transcription polymerase chain reaction (RT–PCR) kit one step cat # BSBO 7 MI for (infectious bronchitis,, TRT and Avian Influenza) as extraneous RNA agents in IBD variant vaccine. Amplification by using Ferments DreamTaq green PCR Master Mix Cat # K 1084 for (ILT, DH, Fowl Pox and Marek's disease virus) as extraneous DNA agents in IBD variant vaccine .The amplicone was subjected to sequencing 4. Haemaulutmation test (HA): (Majujabe and Hitchner ,1977) It carried out according to the standard procedure described by Majujabe and Hitchner(1977) to detect of extraneous haemagglutinating agents inprepared IBD vaccine according to 9 CFR Ch. 1. (1-109) Edition 113 -3 3 (2009). 5. Haemagglutination inhibition (HI) test for NDV: The test was carried out according to the standard procedure described by MajujabeandHitchner (1977) for the haemagglutinating activity of NDV antigen was an essential primary procedure using the HA test (Anon, 1971) to determine HA unites used in HI test 6. Titration of local field isolates in BGM-70 cell lines: This method was carried out according to Pedro and Graham (1980), this technique used for evaluation of the potency of tissue culture adopted live IBDV vaccines. The TCID50was calculated according to Reed and Muench (1938) -3- 7. Evaluation of bursal lesion: a.Organ/body weight ration: .It was carried out according to Sharma et al.,(1989):Collected bursae; spleen;orproventruclus were weighed and the organ/body weight ratio was determined as follows: Organ weight (gm) Organ/body weight ratio= Body weight (gm) b. Estimation of bursal weight index: x 1000 It was carried out according to Lucio and Hitchner(1979). B:BW ratio of the inoculated group Bursal weight index= Mean of B:BW ratio of the non-inoculated control group Chickens with bursal index lower than 0.7 were considered to have bursal atrophy Results: Preparation of living T.C. IBD vaccine from local variant strain: Adaptation of virus in BGM-70: The local variant strain was adapted on BGM-70 cell line and for preparation of living vaccine.In table (1), the results showed that BGM-70 cell line was satisfactory as it yielded good cytopathic changes in BGM-70 cell started after 7 days post inoculation (P.I) in the first passage, while the maximal development of CPE was achieved at 2 days P.I. in the fifth passage. The observed cytopathic changes were characterized by a marked granulation of cell cytoplasm, cell rounding, cell aggregation, loss of adherence of cells and formation of cell syncytia. The IBDV had a titre of 10 7.5 TCID50/ml (104.5 TCID50/ dose) after adaptation on BGM-70 cell culture. Quality control of prepared living variant T.C (IBD) vaccine: Bacterial and fungal sterility: The results of living T.C IBD vaccine is free from bacterial and fungal contamination. Mycoplasmic sterility The results of T.C IBD vaccine is free from mycoplasma Identity: Polymerase chain Reaction (PCR) by using primer specific to infectious bursal virus Visualization of the PCR products on a 1 % agarose gel, DNA band was seen at 300bp . Purity tests: The T.C IBD vaccine does not contain any extraneous agent either haemagglutinating agent inoculation of 9 – 10 day old embryonated SPF eggs; primary culture of chicken embryofibroblasts (Test for avian leucosis virus) or by using PCR test for on other extraneous viral agents. Virus titration: After adaption virus was tittered by serial passages on BGM-70 cell line the virus titer increased in the higher passage than the lower ones as seen in table (1) .The virus titer reached 7.5 log10 TCID50 after five passages in BGM-70 cell line Safety of prepared vaccine: -4- Ten field doses of locally prepared variant T.C. vaccine and imported classical D78 one are administered by eye drop to each of 15 SPF one day old chickens; respectively as groups 1&2 while another 15 SPF chickens were kept as control, all chickens were kept under observation for 3 weeks for any systemic reactions, and post-mortem examinations. Five chickens on each group at7th, 14th day post vaccination. Bursae of Fabricius of chickens were examined macroscopically for evidence of any changes due to IBDV infection All vaccinated chickens had no change or show notable clinical signs or macroscopic lesions that are attributable to the vaccine with comparison the negative control chickens. Safety in mammalian species (Ecotoxicitytest): Thirty Swiss mice were divided into three groups (10/ group) as follow: Group (1): vaccinated group receive the T.C IBD vaccine with 10 x dose intra peritoneal, and group (2): receive imported classical D78; Group (3): non-vaccinated controlled group. T.C IBD vaccine proved to safe for mammalian species as there was no mortality occurred in any group, nor general or local reaction. Growth was identical in the all groups during the period of observation. . Results of safety test in mammalian species (Ecotoxicity lest) gave no evidence for abdominal toxicity in Swiss mice inoculated intra peritoneal with 10 X dose of T.C IBD vaccine. Evaluation of organ/body weight ratio. .Forty SPF chicks were divided into 2groups for studding the effect of the prepared living TCIBD variant vaccine on organs/body weight in relation to control birds. Evaluation of the immunosuppressive effect of the prepared living T.C.IBD vaccine. Fifty SPF one chicks were used for measuring the possible immunosuppression of prepared variant IBD vaccine by measuring HI titer against ND vaccine compared with control one. Potency test and cross protection: Three hundred and fifty SPF one day old chickens were divided into equally 2 groups (A&B) into 7 subgroups as in tables (4). Groups from (1- 4) vaccinated via eye drops with different imported vaccines (CEVA, IZO, IntervetandHipra, respectively) and group (5) was vaccinated with locally prepared T.C variant vaccine. While groups (6&7) kept as control group (6) control +ve for vvIBD, in subgroup (A) and for variant local isolate in subgroup (B) while group 7 control –ve for all experiment. Chickens were kept under observation for 3 weeks post vaccination and the serum samples were collected from all groups. The immune response was determined in vivo by challenging birds with 103.5EID50 / dose virulent IBD strain for all vaccinated groups except group (7) in subgroups (A) and challenged with variant local isolate with dose 103.5EID50 in subgroups (B) and in vitro by measuring ELISA titer post-vaccination and their mean antibody titer that shown in table (4). Detection of protective dose fifty for prepared vaccine: One hundred and twenty five , one day old SPF chicks used for this experiment and divided into (5) groups as following : group 1,2,3and 4vaccinated via eye drop route by full,0.5, 0.25and 0.125 of dose respectively while group 5 kept as positive control . Control negative non-vaccinated non-challenge group of 5 chickens was also included for this experiment as group (6). All groups were kept under observation daily. At 3 weeks post vaccination the serum samples were collected from all groups .The immune response was determined by ELISA test and challenging the 1st five groups with known vvIBDV with titer 103.5 EID50 / dose The calculation of PD50: -5- D = 50 - protection %of dose less than 50% Protection% more than 50%-protection% of dose less than 50% Y = Dose over 50% Dose less than 50 X = d x log y Discussion We designated our trails for the local variant strain preparation of T.C. vaccine compared with living IBD vaccines that used in the field as method of prevention the economic losses and the immunosuppressive effect of subclinical form of IBD. The locally prepared living variant T.C. IBD vaccine on BGM-70 cell gave good results for propagation and titration 7.5 log 10 TCID50 / ml, the harvested and subjected as described by Hassan and Saif (1996) and Hassan et al., (1996) , and our results agreed with the result obtained by Lee and Lukert (1986) as they attempted isolation of IBDV from turkeys and chickens as well as from samples of challenge strains received from other laboratories . Turkey strain (five of five) were readily adapted to CEF cells after 3 to 10 blind passages , only two of nine chicken strains could be grown only in chicken embryo fibroblast cells, and with that of Saif (1984) who used BGM-70 cells successfully for isolation of IBDV from the bursa of naturally infected chickens;.The result of titration was judged according to the parameters of code of Federal Regulation USA "Part 133. 331-9 CFR.1 (2008) in which IBDV titers must be not less than 103.5 TCID50 / dose. So the prepared TC IBD Variant strain was Satisfactory with 104.5 TCID50/dose. Lukert and Davis (1974) who successfully adapted wild type IBDV from infected bursa to grow in cell derived from the chicken embryo bursa which considered as nonimmunosuppressive vaccine.According to the parameters of Code of Federal Regulation, USA, Part 133331-9 CFR Ch. 1, 1-1-97 Ed"(2008) in which IBDV titer must be notless than 3.5 log10 EID50/ml, the intermediate vaccine lost its ability to beprotective by 10 weeks and 12 weeks at 37°C and 25°C ; respectively. While the mild vaccine lost its ability to be a protective vaccine by the 6 th week and 10th week al 37°C and 25°C;respectively. The above mentioned results were agreed with Fekria et al., (1993) who reported a reduction in virus titer equal l0 6.3 log10 EID50 for 6 months at room temperature..The results of sterility, safety, potency and immunogenicity of the local variant IBDV was done according to Federal regulation USA "part 133. 331 – 9CFR ch.1, 1-1-97 ED"(2008); Giambrone and Closser (1990) and Ismail and Saif (1991). It is free from bacterial fungal and Mycoplasma which judged according to parameters of European pharmacopoeia (1998) in which the vaccine must be sterile and free from any contamination. The TCIBDV does not contain any extraneous agent eitherhaemagglutinating agent; avian leucosis virus or any another extraneous, viral agents when use SPF inoculation, tissue calture; ELISA test or PCR. These results was judged according to 9CFR (2009), Europian pharmacopeia (1989 and 1998) , British pharmacopeia (1990) and GRIMV (1992). PCR used for detect of identity of TCIBDV as described in OIE (2010)All experimental chicks had no changes or show notable clinical signs or macroscopic lesions that are attributable to the vaccine in chick’s vaccination with 10X field dose in comparison with the negative control chicks.Theirbursae of Fabricius are examined macroscopically for evidence of any changes due to IBDV. Our results for ecotoxicity test according to European pharmacopeia (1997).So that TCIBD vaccine which locally prepared from variant isolate was safe and sterile. Bursal indexes in vaccinated SPF groups were significantly higher than the challenge controls (table-2). The TCIBD locally prepared variant vaccine protected against bursal damage as indicated by significantly lower bursal lesions in vaccinated birds (Perozo,et al., 2009), the bursae from chickens with bursal/body weight index higher than 0.7 found to be histologically normal (Lucio and Hitchner 1979) -6- Bursa / Body weight ratio were calculated according to Sharma et al., (1989) revealed the results . Assessment of bursal atrophy based on the criteria proposed by Lucio and Hitcliner (1979), that BI equal to or Less than 0.7 to be considered atrophy, the vaccinated group revealed no bursal atrophy during 13 days PI. Variant infected group revealed marked, rapid-decrease in bursal size as early as 36 hours PI up to 13 days PIas recorded in histopathology. Infected group with classic virus strain revealed an increase in bursal size to approximately twice the size of the control group within 4 days PI, then decreased in size to approximately half of the control group during 13 days PIBursal atrophy in this group begin to atrophied on the 6th day PI (BI=0.53) to the end of the observation period. These findings are in agreement with the findings of Rosenberger et al. (1987); and Tsai and Saif (1992) and Lukert and Saif (2008).Chickens infected with local, variant T.C IBDVs had larger splenic size and SI, mostly during the observation periods PI (from 36 hours to 14 days PI) as compared with control ones. These findings are in agreement with Tsai and Saif (1992). According to the assessment criteria proposed by Lucio and Hitchner (1979),that splenic index (SI), lower than the lowest control index considered atrophied, whereas an index higher than the highest control index was considered as hypertrophied. Based on these criteria, all IBDV infected groups showed hypertrophied spleens. These results agreed withthe findings of Resales et al. (1989).These results correlated with histopathological findings for evaluation of the extent of splenic damage, the picture revealed early mild lymphocytic depletion accompanied with mild edema in all infected groups. This finding explain the hypertrophy of spleen and agreed with those reported by Tanimura et al., (1995), who reported the ability of field and vaccinal strains of 1BDV to infect monocytes and macrophages and enlargement of splenic reticular cells.. The locally prepared variant T.CIBD vaccine is non-immunosuppressive as shown in table(3) Our results agree with Mazariegos et al., (1990) who studied the effect of pathogenesis of seven commercially available IBD vaccines and immune-suppressive effects on SPF chicks. These vaccine strains are intermediate in their pathogenicity in susceptible SPF chickens. One-day-old and 3-day-old SPF chickens were vaccinated with these vaccines. Two weeks after IBD vaccination, they were vaccinated with NDV. The pathogenic and immunosuppressive effects of IBD vaccines were evaluated by the antibody response to NDV vaccination. It was found that these strains were highly variable in their virulence and immunosuppressive properties. Three tested strains were found to be highly virulent and immunosuppressive; two others were moderate; and two could be classified as mild At potency test of the prepared local variant vaccine Three hundred and fifty SPF one day old chicks were divided equally 2 subgroups (A&B), into each group (7) subgroups as shown in tables (4). The subgroups (A&B) from 1,2,3 and 4 in each group were vaccinated via eye drops with imported vaccines from ( CEVA, Izo, Intervet and Hipra; respectively) while subgroup(A&B) of no. 5 was vaccinated with locally prepared T.C. variant vaccine. At the same time subgroup 6 and 7 were consider positive and negative control. At 3 weeks of age, serum samples were collected from all subgroups for measuring antibody titer using ELISA. The group (A) was challenged with vvIBD with a dose of 10 3.5EID50 and the group (B) was challenged with variant local isolate with a dose of 103.5EID50.The birds were observed for 2 weeks post challenge and signs, lesions and mortalities were recorded.Cross protection for the prepared vaccine gave protection 96% and 100% against vvIBD and variant one; respectively. GMT ELISA titer 8271 when compared with imported vaccine which give 92,100,100, 96%for intermediate plus, intermediate, 228E and classical D78; respectively with GMT ELISA titer 10317,10610,10251,11092. our results accord withKhafagy et al., (1991) who found that mortalities by three Egyptian vvIBDV isolates in SPF chickens were 40% , 80% and 100% and with Gimbrone and Clay (1986) and Mousa et al., (1988). Amal (1999) showed the ELISA antibody titer was in gradual increase and reached its maximal level at the 3rd week post vaccination and the antibody titer was higher in chicken groups vaccinated with intermediate strain than those with mild strain vaccine. The same results recorded also by Monier (1997).High serologic immune response as early as 36 hours PI with variant, classic and vaccinal strains and gradually increased to high levels at the end of the observation period (49 days of age). These results, -7- are in partial agreement with those reported by Bayyari et al,. (1996), who recorded neutralizing antibodies 3 days PI with standard and variant isolates while at 4 days after vaccination with an intermediate type of vaccine, associated with complete protection after challenge Van den Berg and meulemans( 1991), and mostly attributed to the used kits where the wells of the plates were coated with IBDV-derived bursal homogenates. Finally, protective dose fifty for prepared vaccine was calculated when SPF chicks were inoculated with full, 0.5, 0.25 and 0.125 field dose then challenged with 103.5 EID50/dose of local prepared variant vaccine. The results revealed that only 0.125 and 0.25 does was insignificant PD50, while full & half dose were significant PD50 according to OIE (2008) that the protective dose of vaccine must be more than 90%. From above mentioned results we can use living T.C. IBD vaccine prepared from local variant isolated virus strain as method for control of IBD disease in Egypt. On the other hand; the subclinical for of the disease caused by variant strains need different programmers for control. Inactivated vaccines and live vaccine made from variant strains protected chickens from the disease caused by either variant or standard strains, whereas inactivated vaccines made from standard strains did not protect, or only partially protect against challenge with variant strains (Rosenberger et al., 1985, and Ismail and Saif, 1991). Corresponding author Susan, S. El-Mahdy Central Lab. for Eval. of Vet. Biol. Abbassia Cairo, Egypt (CLEVB) [email protected] References: Allan ,W.H.; Faragher, J.T. and Cullen, G. A. (1972): Immunosuppression by theInfectious bursal agent in chickens immunized by Newcastle disease .Vet. Rec., 90:511-512. Amal,A.S.A.(1999): Effect of infectious bursal disease vaccines on immune response of chickens against Newcastle disease vaccines.M.V.Sc. Thesis, Poultry Diseases, Fac. Vet.Med., Cairo Univ. Anon,A.W.(1971): Method for examing and for identifying and quantifying avian pathogens National Academy of Scince ,Washington, D.C.,270-279. Bayyari, G.R., J.D. Story, J.N. Beasley, and J.K. Skeetes (1996). Pathogenicity studies of an Arkansas variant infectious bursal disease virus. Avian Dis. 40:516-532. Bekhit,A.B.A. (1996): Very virulent form infectious bursal disease in Egypt. 3rd Vet, Med. Cong. Zagazig. British pharmacopoeia (1990) (UK) MAL 74 and 79 testing of virus purity. Code of American Federal Regulation (2009): (9CFR).Published by office of the federal register national archives records service, Animals and animal products Ch.1 (1-1-09 Edition 11.30-113-33. Code of Federal Regulation USA (2008): Part 133.331-9 CFR Ch. 1.1-1-97 Ed. Cosgrove, A.S.(1962): An apparently new disease of chickens-avian nephrosis, Avian Dis., 6:385:389 Council of Europe (1999). 2.6.3 Avian viral vaccines: Tests for extraneous Agents in seed lots pharmeuropa 11, 188-191. El-Batrawi, A.M.; and El-Kady, M.F. (1990): Studies on sever outbreaks of infectious bursal disease 3-determination of the criticalage of susceptigilty in maternally immune chicks. Proc. Of 2nd Sci. Conf. Egypt. Vet. Poult. 264 – 269. El-Khiat,F. (2003): Evaluation of some vaccination programmes against infectious bursal disease virus in Egypt. Ph. D. Thesis Fac.Vet. Med. Kafr-Elshikhbranch,Tanta Univ. El-Sergany H.A.; Ann, Moursi.; Saber, M.S.andMohammed,M.A.(1974): A preliminary investigation on the occuarance of Gumboro disease in Egypt.Egypt.j.Vet. Sci., 11-17. European Pharmacopaeia (1997): Studying safety test (Control test technique) Monographs 0442, 0450 and 1068. European pharmacopaeia, 2ndadition (1989): V2.1.3.5. using ELISA test for testing viral purity. European Pharmacopaeia, 3 rd edition supplement (1998): 2.6.1 (Bacterial and fungal sterility) 2.6.5 Test for extraneous virus using cell culture 2.6.7 Mycoplasmas, culture method -8- Fekria A. El-Bordeny; Salwa, A. El-Assily; Ensaf, M. Khashabah; Gergis, S.M. and Nadia M. Hassan (1993): Some studies on vaccinal strain of infectious bursal disease .Proc. 42th West Poult Dis. Conf. California Feb. 28. Giambrane, J.J. and Classer, J. (1990): Efficacy of live vaccines against serologic subtypes of infectious bursal disease virus.Avian Dis., 34 : 7-11. Giambrone, J.J. and Clay, R.p. (1986): Evaluation of the immunogenicity , stability, pathogenicity and immunodepressive potential of four commercial live infectious bursal disease vaccine.Poultry Science, 65(7): 1287-1290. GRIMV (1992): General Requirements for the production and control of bacterial and viral vaccines for veterinary use: 7 BIM 2a 23-32. Hank , J.H. and Wallance ,R.E.(1949): Relation of oxygen and temperature in the preservation of tissue by refrigeration. Proc. Soc. Exptl. Bio. Med., 71:196-200 Hassan, M.K. and Saif, Y.M. (1996): Influence of host system on the pathogenicity, immunogenicity and antigenicity of infectious bursaldisease.Avian Dis., 40: 553-561. Hassan,M.K.; Al-Natour,M.Q.; Ward, L.A, and Saif, Y.M. 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Med., Athens, Georgia,USA. - 11 - Table (1) Propagation and titration of virus on BGM-70 cell lines showing number of passages and time needed appearance of CPE No. of passage Time needed for CPE Virus titre 1 7 days 2.5 2 6 days 3.0 3 5 days 4.5 4 3 days 6.5 5 2 days 7.5 No. : NumberCPE:cytopathiceffect : titre of virus (log 10 TCID50 / ml) Table (2) Evaluation of organ/body weight ratio: No fo Treated SPF Chicks 3 days O days 7 days 10 days 14 days B S P B S P B S P B S P Vairant ICIBD 20 0 *1.5 *1.3 1.0 1.4 *1.2 0.95 0.7 *0.49 1.0 0.66 0.78 0.9 Control 20 0 1 1 1 1 1 1 1 1 1 1 1 1 B: bursal/body weight ratio S: spleen /body weight ratioP: proventriculus/body weight ratio *significant difference at p≤ 0.05 - 12 - Table (3): Results of immunosuppression test No. of group G1 vaccinated G2 Indicator G3 control +ve G4 control -ve No. of dead birds/ total number of birds 2/15 Protection percentage Mean HI log2 titer 86.6% 6.9 1/15 93.03% 7.2 15/15 0% 1.9 0/5 100% 1.9 The experimental chicks were fed on sterile ration and water, divided into 4 groups as follow: G1: 15 birds vaccinated with prepared living variant T.C. vaccine at one day old via eye drop and then at 2 weeks old vaccinated with ND vaccine HB1 with titer 107.5 EID50 / dose by eye drop. G2: 15 birds vaccinated with ND vaccine HB1 at 2 weeks old and act as indicator for NDV. G3: 15 birds control non-vaccinated challenge group with vvNDV with titer 106.5 EID50 / dose. G4: 5 birds control non-vaccinated non-challenge. All groups measuring the immune response by HI test at 4 weeks old. The locally prepared living attenuated T.C. variantstrain is consider as nonimmunosuppressivevaccine - 13 - Table (4): Results of potency test: No. of groups Types of vaccine used GMT of ELISA No. of dead birds/ total number of birds Protective % * G1(A&B) Vac1 10317 2/25 92% G2(A&B) G3(A&B) G4(A&B) Vac2 Vac3 Vac4 10610 10251 11092 0/25 0/25 1/25 100% 100% 96% G5(A) G5(B) G6(A) G6(B) T.C.V.V T.C.V.V Control+ve Control+ve 8271 8271 156 156 2/25 0/25 20/20 5/20 96% 100% 0% 80% G7(A&B) Control-ve 156 0/10 100% N.B.: GMT of ELISA titer of control positive serum is equal or more than 3000 GMT: Geometric mean of ELISA antibody titer against IBDV. A:vaccinated birds challenged with vvIBDv B:vaccinated birds challenged with isolated variant IBD strain Vac 1: intermediate plus CEVA vaccine with titer 105.5 EID50 / dose Vac2: intermediate (gumboro 2) IZO with titer 105.5 EID50 / dose. Vac3: intermediate Nobilis 228E with titer 105.5 EID50 / dose. Vac4: classical D78 Hipra with titer 105.2 EID50 / dose. T.C.V.V.: locally prepared T.C. variant vaccine with titer 104.5 EID50 / dose and challenged with field isolated vvIBDV in subgroup (A) andsubgroup (B) challenged with field isolated variant IBDstrain. G6: non vaccinated and challenged with vvIBDV in subgroup(A)and subgroup(B) challenged with field isolated variant IBD strain G7 (A&B): non vaccinated and non-challenged group No clinical sings or lesion were recorded in all vaccinated group. Birds in group (6) showed atrophied bursae with slight hemorrhages on proventriculus. *Significant difference at P ≤ 0.05. - 14 - Table (5): Results of PD50 of different doses for locally prepared T.C.IBD variant vaccine: Dose No. of group ELISAGMT Protection against challenge AntibodyTiter Total no. of birds No. of protect bird No. of dead bird Protect % Full dose G1 8271 25 23 2 92 0.5 dose G2 8007 25 23 2 92 0.25 dose G3 2610 25 22 3 88 0.125 dose G4 2040 25 20 5 80* Control+ve G5 242 25 0 25 0 Control–ve G6 242 5 5 0 100 N.B.: the protective dose for vaccine must be more than 90% according to OIE (2008). GMT of ELISA titer of control positive serum is equal or more than 3000. GMT = Geometric mean of ELISA antibody titer against IBDV. *significant difference at p≤0.0 - 15 -